The invention provides nucleotide sequences of coryneform bacteria which code for the alr gene, a host-vector system for coryneform bacteria using the alr gene, processes for the preparation of chemical compounds using the host-vector system and processes for the preparation of D-amino acids, in particular D-alanine or D-valine, using coryneform bacteria or Enterobacteriaceae in which the alr gene of coryneform bacteria is present in enhanced form. All references cited herein are expressly incorporated by reference. Incorporation by reference is also designated by the term “I.B.R.” following any citation.
Chemical compounds, which means, in particular, L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids, are used in human medicine, in the pharmaceuticals industry, in cosmetics, in the foodstuffs industry and in animal nutrition.
Numerous of these compounds are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or the working up to the product form by, for example, ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites or are auxotrophic for metabolites of regulatory importance and which produce the particular compounds are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains, by amplifying individual biosynthesis genes and investigating the effect on production.
Naturally occurring plasmids and plasmid vectors prepared from these are an important prerequisite for improving the production properties of coryneform bacteria. The construction of plasmid vectors for this group of industrially important bacteria is substantially based on cryptic plasmids which are equipped with suitable antibiotic resistance markers capable of functioning in Corynebacteria or Brevibacteria (U.S. Pat. No. 5,158,891 I.B.R. and U.S. Pat. No. 4,500,640 I.B.R.). These plasmid vectors can be employed for cloning and enhancing genes which participate in the production of chemical compounds, such as, for example, L-amino acids, vitamins or nucleosides and nucleotides. Production of the desired substances can be influenced in a positive manner by expression of the particular genes. Thus e.g. cloning of a DNA fragment which codes a protein for a lysine exporter led to an improvement in the fermentative production of L-lysine with Corynebacterium glutamicum strain MH20-22B (DE-A 19548222 I.B.R.).
In contrast to the known bacterium of equal industrial importance Escherichia coli, only a limited number of natural plasmids and suitable selection markers for the development of cloning and expression vectors are known for Corynebacteria and Brevibacteria, in particular Corynebacterium glutamicum. Selection systems have hitherto been available only in the form of two antibiotic resistance markers which have been identified on the streptomycin/spectinomycin resistance plasmid pCG4 from Corynebacterium glutamicum ATCC31830 (U.S. Pat. No. 4,489,160 I.B.R.) and on the tetracycline resistance plasmid pAG1 from Corynebacterium melassecola 22243 (U.S. Pat. No. 5,158,891 I.B.R.). Plasmid pCG4 furthermore carries the sulI gene, which imparts sulfamethoxazole resistance and the sequence of which was determined by Nesvera et al. (FEMS Microbiology Letters 169, 391-395 (1998) I.B.R.).
For rapid investigation and improvement of strains which produce the compounds mentioned, it is important to have plasmid vectors which are compatible with one another and have a sufficiently high stability, such as e.g. the plasmid pGA1 from Corynebacterium glutamicum LP-6 (U.S. Pat. No. 5,175,108 I.B.R.). The plasmid vectors conventionally employed are composed of components which originate from the species Corynebacterium glutamicum and another species of bacteria, typically Escherichia coli. Foreign DNA is introduced into the species Corynebacterium glutamicum by this procedure. Stable plasmid vectors which are capable of functioning and contain only species-characteristic DNA with an antibiotic-free selection possibility and therefore meet the criteria of self-cloning are not known to experts.
Processes for the preparation of D-amino acids with Corynebacterium glutamicum by fermentative or biocatalytic methods are not known to experts.